Cancer is one of the leading causes of death worldwide, and lung carcinoma is the leading cause of cancer-related death. Lung cancer is classified histologically as either small cell lung carcinoma (SCLC, ~15% of lung cancers) or non-small cell lung carcinoma (NSCLC, ~85% of lung cancers). Genetic alterations driving NSCLC are known in only ~55-60% of cases, and are mainly attributable to mutually exclusive mutations in the KRAS, EGFR, ALK, HER2, BRAF, PI3KCA, MET, AKT1, or MAP2K1 genes. Clinical trials have shown that targeted therapy using tyrosine kinase inhibitors (TKIs) against mutant EGFR or ALK proteins is superior to traditional chemotherapy, which non-selectively kills rapidly dividing cells. These findings highlight the value of: 1) designing effective inhibitors of other drivers of NSCLC, 2) the discovery of other driver mutations, and 3) the development of companion diagnostics. Chromosomal rearrangements in the ROS1, ALK, and RET gene account for ~7% of NSCLC cases, equating to ~140,000 new annual cases of NSCLC worldwide. Constitutive activation of the ROS1 tyrosine kinase domain is inhibitable by the FDA-approved ALK inhibitor crizotinib, which has shown efficacy in clinical trials with patients with ROS1-rearranged NSCLC. As a consequence of targeted therapy, however, secondary drug-resistance mutations in ROS1 and ALK have arisen in patients no longer responding to therapy. Some, but not all, secondary mutations in ROS1 or ALK can be overcome with alternative TKIs, such as cabozantinib, foretinib, or ceritinib. Thus, knowing the status of ROS1, ALK, or RET mutations can inform both first and second line therapy. GeneTAG Technology, Inc. specializes in developing DNA Detection Switch (DDS) probe systems for real-time PCR that use labeled probes and competitive, quencher-labeled antiprobes. Our novel probe systems offer unparalleled single-based discrimination (iDDS probes), error-checking amplification (ZIPR probes), or cost-effective, multi-target screening with generic components (Universal probes). The Specific Aims of this Phase I application are 1) to develop a two-stage screening assay for ROS1, ALK, and RET gene fusions, and 2) to develop iDDS probe endpoint detection assays against drug-resistance mutations in ROS1 and ALK. Performing endpoint analysis of amplified targets increases the diagnostic yield obtainable from small samples, such as fine-needle aspirates. Experiments will be performed with synthetic DNA ultramers; cDNA from cells overexpressing the fusion templates; and deidentified, blinded FFPE tissue sections from NSCLC patients from collaborators at Emory Hospital and Akesogen. Successful completion of this proposal will justify subsequent Phase II validation studies in preparation for filing for FDA approval. Currently, no FDA- approved diagnostics assays are available to detect ROS1 or RET rearrangements, or drug-resistance mutations in ROS1 or ALK. Thus, the proposed studies address an important unmet need in patient care.